1887

Microbiology Society logo

Volume 147, Issue 1

LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk Free

Abstract

Amino acid aminotransferases (ATases), which catalyse the last biosynthetic step of many amino acids, may have important physiological functions in during growth in milk. In this study, the aspartate ATase gene () from LM0230 was cloned by complementation into DL39. One chromosomal fragment putatively encoding was partially sequenced. A 1179 bp ORF was identified which could encode for a 393 aa, 432 kDa protein. The deduced amino acid sequence had high identity to other AspC sequences in GenBank and is a member of the Iγ family of ATases. Substrate-specificity studies suggested that the lactococcal AspC has ATase activity only with aspartic acid (Asp). An internal deletion was introduced into the chromosomal copy of by homologous recombination. The wild-type and mutant strain grew similarly in defined media containing all 20 amino acids and did not grow in minimal media unless supplemented with asparagine (Asn). The mutant strain was also unable to grow in or significantly acidify milk unless supplemented with Asp or Asn. These results suggest that only one lactococcal ATase is involved in the conversion of oxaloacetate to Asp, and Asp biosynthesis is required for the growth of LM0230 in milk.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): amino acid biosynthesis , Ap, ampicillin , aspartate aminotransferase , aspartate biosynthesis , ATase, aminotransferase , CFE, cell-free extract , Em, erythromycin , lactic acid bacteria , Lactococcus lactis and Opp, Lactococcus lactis oligopeptide transport system
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-1-215
2001-01-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/1/1470215a.html?itemId=/content/journal/micro/10.1099/00221287-147-1-215&mimeType=html&fmt=ahah

References

  1. Atiles, M. W., Dudley, E. G. & Steele, J. L.(2000). Gene cloning, sequencing, and inactivation of the branched-chain aminotransferase of Lactococcus lactis LM0230. Appl Environ Microbiol 66, 2325-2329.[CrossRef] [Google Scholar]
  2. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.[CrossRef] [Google Scholar]
  3. Ausubel, F. M., Brent, R., Kingston, R. E., More, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1989).Current Protocols in Molecular Biology, vol. 1. New York: Green Publishing Associates and Wiley Interscience.
  4. Bartsch, K., Schneider, R. & Schulz, A.(1996). Stereospecific production of the herbicide phosphinothricin (glufosinate): purification of aspartate transaminase from Bacillus stearothermophilus, cloning of the corresponding gene, aspC, and application in a coupled transaminase process. Appl Environ Microbiol 62, 3794-3799. [Google Scholar]
  5. Biswas, I., Gruss, A., Ehrlich, S. D. & Maguin, E.(1993). High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175, 3628-3635. [Google Scholar]
  6. Bruinenberg, P. G., Vos, P. & de Vos, W. M.(1992). Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk. Appl Environ Microbiol 58, 78-84. [Google Scholar]
  7. Chen, Y. & Steele, J. L.(1998). Genetic characterization and physiological role of endopeptidase O from Lactobacillus helveticus CNRZ32. Appl Environ Microbiol 64, 3411-3415. [Google Scholar]
  8. Chopin, A.(1993). Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria. FEMS Microbiol Rev 12, 21-38.[CrossRef] [Google Scholar]
  9. Christensen, J. E., Dudley, E. G., Pederson, J. A. & Steele, J. L.(1999). Peptidases and amino acid catabolism in lactic acid bacteria. Antonie Leeuwenhoek 76, 217-246.[CrossRef] [Google Scholar]
  10. Clarke, L. & Carbon, J.(1976). A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome. Cell 9, 91-99.[CrossRef] [Google Scholar]
  11. Cocaign-Bousquet, M., Garrigues, C., Novak, L., Lindley, N. D. & Loubiere, P.(1995). Rational development of a simple synthetic medium for sustained growth of Lactococcus lactis. J Appl Bacteriol 79, 108-116.[CrossRef] [Google Scholar]
  12. Davidson, B. E., Kordias, N., Dobos, M. & Hillier, A. J.(1996). Genomic organization of lactic acid bacteria. Antonie Leeuwenhoek 70, 161-183.[CrossRef] [Google Scholar]
  13. Dickely, F., Nilsson, D., Hansen, E. B. & Johansen, E.(1995). Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector. Mol Microbiol 15, 839-847.[CrossRef] [Google Scholar]
  14. Efstathiou, J. D. & McKay, L. L.(1976). Inorganic salts resistance associated with a lactose-fermenting plasmid in Streptococcus lactis. J Bacteriol 130, 257-265. [Google Scholar]
  15. Foucaud, C., Kunji, E. R., Hagting, A., Richard, J., Konings, W. N., Desmazeaud, M. & Poolman, B.(1995). Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides. J Bacteriol 177, 4652-4657. [Google Scholar]
  16. Gao, S. & Steele, J. L.(1998). Purification and characterization of oligomeric species of an aromatic amino acid aminotransferase from Lactococcus lactis subsp. lactis S3. J Food Biochem 22, 197-211.[CrossRef] [Google Scholar]
  17. Gao, S., Oh, D. H., Broadbent, J. R., Johnson, M. E., Weimer, B. C. & Steele, J. L.(1997). Aromatic amino acid catabolism by lactococci. Lait 77, 371-381.[CrossRef] [Google Scholar]
  18. Gao, S., Mooberry, E. S. & Steele, J. L.(1998). Use of 13C nuclear magnetic resonance and gas chromatography to examine methionine catabolism by lactococci. Appl Environ Microbiol 64, 4670-4675. [Google Scholar]
  19. Gerhardt, P., Murray, R. G. E., Woods, W. A. & Krieg, N. R. (1994).Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
  20. Hagting, A., Kunji, E. R., Leenhouts, K. J., Poolman, B. & Konings, W. N.(1994). The di- and tripeptide transport protein of Lactococcus lactis: a new type of bacterial peptide transporter. J Biol Chem 269, 11391-11399. [Google Scholar]
  21. Helinck, S., Richard, J. & Juillard, V.(1997). The effects of adding lactococcal proteinase on the growth rate of Lactococcus lactis in milk depend on the type of enzyme. Appl Environ Microbiol 63, 2124-2130. [Google Scholar]
  22. Hillier, A. J., Rice, G. H. & Jago, G. R.(1978). Metabolism of [14C]bicarbonate by Streptococcus lactis: the synthesis, uptake and excretion of aspartate by resting cells. J Dairy Res 45, 241-246.[CrossRef] [Google Scholar]
  23. Hofmann, K., Bucher, P., Falquet, L. & Bairoch, A.(1999). The PROSITE database, its status in 1999. Nucleic Acids Res 27, 215-219.[CrossRef] [Google Scholar]
  24. Holo, H. & Nes, I. F.(1989). High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55, 3119-3123. [Google Scholar]
  25. Jensen, R. A. & Calhoun, D. H.(1981). Intracellular roles of microbial aminotransferases: overlap enzymes across different biochemical pathways. Crit Rev Microbiol 8, 229-266.[CrossRef] [Google Scholar]
  26. Jensen, R. A. & Gu, W.(1996). Evolutionary recruitment of biochemically specialized subdivisions of family I within the protein superfamily of aminotransferases. J Bacteriol 178, 2161-2171. [Google Scholar]
  27. Juillard, V., Le Bars, D., Dunji, E. R. S., Konings, W. N., Gripon, J.-C. & Richard, J.(1995a). Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk. Appl Environ Microbiol 61, 3024-3030. [Google Scholar]
  28. Juillard, V., Laan, H., Kunji, E. R., Jeronimus-Stratingh, C. M., Bruins, A. P. & Konings, W. N.(1995b). The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides. J Bacteriol 177, 3472-3478. [Google Scholar]
  29. Katayama, M., Sakai, Y., Okamoto, S., Ihara, F., Nihira, T. & Yamada, Y.(1996). Gene organization in the ada–rplL region of Streptomyces virginiae. Gene 171, 135-136.[CrossRef] [Google Scholar]
  30. Konings, W. N., Poolman, B. & Driessen, A. J. M.(1989). Bioenergetics and solute transport in lactococci. Crit Rev Microbiol 16, 419-476.[CrossRef] [Google Scholar]
  31. Kunji, E. R. S., Fang, G., Jeronimus-Stratingh, C. M., Bruins, A. P., Poolman, B. & Konings, W. N.(1998). Reconstruction of the proteolytic pathway for use of β-casein by Lactococcus lactis. Mol Microbiol 27, 1107-1118.[CrossRef] [Google Scholar]
  32. LeMaster, D. M. & Richards, F. M.(1988). NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry 27, 142-150.[CrossRef] [Google Scholar]
  33. McKay, L. L., Cords, B. R. & Baldwin, K. A.(1973). Transduction of lactose metabolism in Streptococcus lactis C2. J Bacteriol 115, 810-815. [Google Scholar]
  34. Marino, G., Nitti, G., Arnone, M. I., Sannia, G., Gambacorta, A. & De Rosa, M.(1988). Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus. J Biol Chem 263, 12305-12309. [Google Scholar]
  35. Nakai, T., Okada, K., Akutsu, S., Miyahara, I., Kawaguchi, S., Kato, R., Kuramitsu, S. & Hirotsu, K.(1999). Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate. Biochemistry 38, 2413-2424.[CrossRef] [Google Scholar]
  36. Okamoto, A., Kato, R., Masui, R., Yamagishi, A., Oshima, T. & Kuramitsu, S.(1996). An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8. J Biochem 119, 135-144.[CrossRef] [Google Scholar]
  37. O’Sullivan, D. J. & Klaenhammer, T. R.(1993). High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 137, 227-231.[CrossRef] [Google Scholar]
  38. Poolman, B.(1993). Energy transduction in lactic acid bacteria. FEMS Microbiol Rev 12, 125-148.[CrossRef] [Google Scholar]
  39. Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. & Hopwood, D. A.(1996). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21, 77-96.[CrossRef] [Google Scholar]
  40. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  41. Sorokin, A., Azevedo, V., Zumstein, E., Galleron, N., Ehrlich, S. D. & Serror, P.(1996). Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome. Microbiology 142, 2005-2016.[CrossRef] [Google Scholar]
  42. Sung, M.-H., Tanizawa, K., Tanaka, H., Kuramitsu, S., Kagamiyama, H. & Soda, K.(1990). Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species. J Bacteriol 172, 1345-1351. [Google Scholar]
  43. Sung, M.-H., Tanizawa, K., Tanaka, H., Kuramitsu, S., Kagamiyama, H., Hirotsu, K., Okamoto, A., Higuchi, T. & Soda, K.(1991). Thermostable aspartate aminotransferase from a thermophilic Bacillus species: gene cloning, sequence determination, and preliminary X-ray characterization. J Biol Chem 266, 2567-2572. [Google Scholar]
  44. Swaisgood, H. E.(1985). Characteristics of edible fluids of animal origin: milk. In Food Chemistry , pp. 791-827. Edited by O. R. Fennema. New York:Marcel Dekker.
  45. Terzaghi, B. E. & Sandine, W. E.(1975). Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29, 807-813. [Google Scholar]
  46. Tinoco, I., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, O. C., Crothers, D. M. & Gralla, J.(1973). Improved estimation of secondary structure in ribonucleic acids. Nat New Biol 246, 40-41.[CrossRef] [Google Scholar]
  47. Tynkkynen, S., Buist, G., Kunji, E., Kok, J., Poolman, B., Venema, G. & Haandrikman, A.(1993). Genetic and biochemical characterization of the oligopeptide transport system of Lactococcus lactis. J Bacteriol 175, 7523-7532. [Google Scholar]
  48. Urbach, G.(1995). Contribution of lactic acid bacteria to flavour compound formation in dairy products. Int Dairy J 5, 877-903.[CrossRef] [Google Scholar]
  49. Wang, H., Yu, W., Coolbear, T., O’Sullivan, D. & McKay, L. L.(1998). A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation. Appl Environ Microbiol 64, 1673-1679. [Google Scholar]
  50. Wang, H., O’Sullivan, D. J., Baldwin, K. A. & McKay, L. L.(2000). Cloning, sequencing, and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2. Appl Environ Microbiol 66, 1223-1227.[CrossRef] [Google Scholar]
  51. Xing, R. & Whitman, W. B.(1992). Characterization of amino acid aminotransferases of Methanococcus aeolicus. J Bacteriol 174, 541-548. [Google Scholar]
  52. Yu, W., Gilles, K., Kondo, J. K., Broadbent, J. R. & McKay, L. L.(1996). Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation. Plasmid 35, 145-155.[CrossRef] [Google Scholar]
  53. Yvon, M., Thirouin, S., Rijnen, L., Fromentier, D. & Gripon, J. C.(1997). An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds. Appl Environ Microbiol 63, 414-419. [Google Scholar]
  54. Yvon, M., Chambellon, E., Bolotin, A. & Roudot-Algaron, F.(2000). Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCDO 763. Appl Environ Microbiol 66, 571-577.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-1-215
Loading
Lactococcus lactis LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk
Microbiology 147, 215 (2001); https://doi.org/10.1099/00221287-147-1-215
/content/journal/micro/10.1099/00221287-147-1-215
/content/journal/micro/10.1099/00221287-147-1-215
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error